PPARβ/δ Regulates Glucocorticoid- and Sepsis-Induced FOXO1 Activation and Muscle Wasting

FOXO1 is involved in glucocorticoid- and sepsis-induced muscle wasting, in part reflecting regulation of atrogin-1 and MuRF1. Mechanisms influencing FOXO1 expression in muscle wasting are poorly understood. We hypothesized that the transcription factor peroxisome proliferator-activated receptor β/δ (PPARβ/δ) upregulates muscle FOXO1 expression and activity with a downstream upregulation of atrogin-1 and MuRF1 expression during sepsis and glucocorticoid treatment and that inhibition of PPARβ/δ activity can prevent muscle wasting. We found that activation of PPARβ/δ in cultured myotubes increased FOXO1 activity, atrogin-1 and MuRF1 expression, protein degradation and myotube atrophy. Treatment of myotubes with dexamethasone increased PPARβ/δ expression and activity. Dexamethasone-induced FOXO1 activation and atrogin-1 and MuRF1 expression, protein degradation, and myotube atrophy were inhibited by PPARβ/δ blocker or siRNA. Importantly, muscle wasting induced in rats by dexamethasone or sepsis was prevented by treatment with a PPARβ/δ inhibitor. The present results suggest that PPARβ/δ regulates FOXO1 activation in glucocorticoid- and sepsis-induced muscle wasting and that treatment with a PPARβ/δ inhibitor may ameliorate loss of muscle mass in these conditions

Del Nido Cardioplegia can be safely administered in high-risk coronary artery bypass grafting surgery after acute myocardial infarction: a propensity matched comparison

Del Nido (DN) cardioplegia solution provides a depolarized hyperkalemic arrest lasting up to 60 minutes, and the addition of lidocaine may limit intracellular calcium influx. Single-dose DN cardioplegia solution may offer an alternative myocardial protection strategy to multi-dose cold whole blood (WB) cardioplegia following acute myocardial infarction (AMI). We retrospectively reviewed 88 consecutive patients with AMI undergoing coronary artery bypass (CABG) surgery with cardioplegic arrest between June 2010 to June 2012. Patients exclusively received WB (n = 40, June 2010-July 2011) or DN (n = 48, August 2011-June 2012) cardioplegia. Preoperative and postoperative data were retrospectively reviewed and compared using propensity scoring. No significant difference in age, maximum preoperative serum troponin level, ejection fraction, and STS score was present between DN and WB. A single cardioplegia dose was given in 41 DN vs. 0 WB patients (p < 0.001), and retrograde cardioplegia was used 10 DN vs. 31 WB patients (p < 0.001). Mean cardiopulmonary bypass and cross clamp times were significantly shorter in the DN group versus WB group. Tranfusion rate, length of stay, intra-aortic balloon pump requirement, post-operative inotropic support, and 30-day mortality was no different between groups. One patient in the WB group required a mechanical support due to profound cardiogenic shock. DN cardioplegia may provide equivalent myocardial protection to existing cardioplegia without negative inotropic effects in the setting of acute myocardial infarction

CaMKII activity is reduced in skeletal muscle during sepsis

Exercise-induced muscle hypertrophy is associated with increased calcium/calmodulin-dependent protein kinase II (CaMKII) expression and activity. In contrast, the influence of muscle atrophy-related conditions on CaMKII is poorly understood. Here, we tested the hypothesis that sepsis-induced muscle wasting is associated with reduced CaMKII expression and activity. Sepsis, induced by cecal ligation and puncture in rats, and treatment of rats with TNFα, resulted in reduced total CaMKII activity in skeletal muscle whereas autonomous CaMKII activity was unaffected. The expression of CaMKIIδ, but not β and γ, was reduced in septic muscle. In additional experiments, treatment of cultured myotubes with TNFα resulted in reduced total CaMKII activity and decreased levels of phosphorylated glycogen synthase kinase (GSK)-3β, a downstream target of CaMKII. The present results suggest that sepsis-induced muscle wasting is associated with reduced CaMKII activity and that TNFα may be involved in the regulation of CaMKII activity in skeletal muscle. Decreased phosphorylation (consistent with activation) of GSK-3β may be a consequence of reduced CaMKII activity, indicating that inhibited CaMKII activity may be involved in the catabolic response to sepsis. J. Cell. Biochem. 114: 1294-1305, 2013. © 2012 Wiley Periodicals, Inc. Copyright © 2012 Wiley Periodicals, Inc

β-Hydroxy-β-methylbutyrate (HMB) prevents dexamethasone-induced myotube atrophy

High levels of glucocorticoids result in muscle wasting and weakness. β-hydroxy-β-methylbutyrate (HMB) attenuates the loss of muscle mass in various catabolic conditions but the influence of HMB on glucocorticoid-induced muscle atrophy is not known. We tested the hypothesis that HMB prevents dexamethasone-induced atrophy in cultured myotubes. Treatment of cultured L6 myotubes with dexamethasone resulted in increased protein degradation and expression of atrogin-1 and MuRF1, decreased protein synthesis and reduced myotube size. All of these effects of dexamethasone were attenuated by HMB. Additional experiments provided evidence that the inhibitory effects of HMB on dexamethasone-induced increase in protein degradation and decrease in protein synthesis were regulated by p38/MAPK- and PI3K/Akt-dependent cell signaling, respectively. The present results suggest that glucocorticoid-induced muscle wasting can be prevented by HMB. © 2012 Elsevier Inc

Treatment of rats with GSK0660 prevents dexamethasone-induced muscle wasting.

<p><i>A)</i> and <i>B</i>) Serum ALT and AST levels in control and dexamethasone-treated (10 mg/kg) rats that were administered vehicle or GSK0660 (1 mg/kg). <i>C</i>) PPARβ/δ activity in EDL muscles from control and dexamethasone-treated rats that were administered vehicle or GSK0660. <i>D</i>) Change of body weight in control and dexamethasone-treated rats that were administered vehicle or GSK0660. <i>E</i>) Muscle weight in control and dexamethasone-treated rats that were administered vehicle or GSK0660. <i>F-H</i>) mRNA and <i>I-K</i>) protein levels for FOXO1, atrogin-1, and MuRF1 in muscles from control and dexamethasone-treated rats that were administered vehicle or GSK0660. Results are means ± SEM with n = 8/group. *<0.05 vs appropriate control group; #p<0.05 vs Veh/Dex group by ANOVA.</p

Dexamethasone increases PPARβ/δ activity in cultured L6 myotubes through a GR-dependent mechanism.

<p><i>A</i>) PPARβ/δ activity in myotubes treated for 24 h with 1 µM dexamethasone or vehicle (Ctr). Results are expressed as percent of control. <i>B</i>) PPARβ/δ protein levels determined by Western blotting in total cell lysates from control and dexamethasone-treated myotubes. Representative Western blots are shown in the upper panel and densitometric quantifications in the lower panel. α-Tubulin was determined for loading control. <i>C</i>) PPARβ/δ activity in control and dexamethasone-treated myotubes. Myotubes were treated for 24 h with dexamethasone (1 µM) in the absence or presence of 10 µM RU38486. <i>D</i>) PPARβ/δ-GR interaction determined by co-immunoprecipitation of myotube proteins extracted from whole cell lysates after treatment of the myotubes for 24 h with 1 µM dexamethasone or vehicle (Ctr). A PPARβ/δ antibody was used for pull-down followed by immuno blotting with a GR or PPARβ/δ antibody. Representative immunoblots are shown in the upper panel and densitometric quantifications of the upper bands in the immunoblts are shown in the lower panel. Results were calculated as percent of control and are means ± SEM with n = 7 or 8/group. *p<0.05 vs Ctr by Student’s t-test (panel A and D) or ANOVA (panel C).</p

Sepsis-induced muscle wasting in rats is improved by treatment with GSK0660.

<p><i>A</i>) PPARβ/δ activity in EDL muscles from sham-operated (SH) and septic (CLP) rats treated with vehicle or GSK0660 (1 mg/kg). <i>B</i>) Muscle weight in sham-operated and septic rats treated with vehicle or GSK0660. <i>C-E</i>) mRNA and <i>F-H</i>) protein levels for FOXO1, atrogin-1, and MuRF1 in muscles from sham-operated and septic rats treated with vehicle or GSK0660. <i>I)</i> GR protein levels in EDL muscles from sham-operated and septic rats treated with vehicle or GSK0660. Results are means ± SEM with n = 8/group except in panel I where n = 4/group. *p<0.05 vs appropriate sham group; #p<0.05 vs Veh/CLP by ANOVA.</p

The PPARβ/δ agonist GW0742 increases protein degradation and induces atrophy of cultured L6 myotubes.

<p><i>A</i>) Protein degradation (% per 24 h) in myotubes treated for 24 h with 0.1 µM GW0742 or vehicle (Ctr). <i>B</i>) Morphology of cultured L6 myotubes after treatment for 24 h with 0.1 µM GW0742 or vehicle (Ctr). Myotubes were photographed under a phase contrast microscope at 100× magnification. <i>C</i>) Diameter of cultured myotubes after treatment for 24 h with 0.1 µM GW0742 or vehicle (Ctr). Results are means ± SEM with n = 8/group.*p<0.05 vs control.</p

Downregulation of PPARβ/δ expression reduces FOXO1 and MuRF1 expression and protein degradation in dexamethasone-treated myotubes.

<p><i>A</i>) mRNA levels for PPARβ/δ and PPARα in myotubes transfected with non-targeting (NT) or PPARβ/δ siRNA. <i>B</i>) PPARβ/δ protein levels in myotubes transfected with non-targeting (NT) or PPARβ/δ siRNA. Representative Western blots are shown in the upper and densitomtric quantifications in the lower panel. <i>C</i>) PPARβ/δ activity in myotubes transfected with non-targeting (NT) or PPARβ/δ siRNA. <i>D-F</i>) Dexamethasone-induced changes in FOXO1, MuRF1, and atrogin-1 mRNA in myotubes transfected with non-targeting (NT) or PPARβ/δ siRNA. <i>G</i>) Protein degradation in myotubes treated for 24 h with or without 1 µM dexamethasone following transfection with non-targeting (NT) or PPARβ/δ siRNA. Results are reported as arbitrary units (AU) or percent of control and are means ± SEM with n = 7 or 8/group. *p<0.05 vs appropriate control group by ANOVA (panel G) or Student’s t-test.</p

PPARβ/δ activation upregulates atrogin-1 and MuRF1 expression through a FOXO1-dependent mechanism in L6 myotubes.

<p><i>A</i>) PPARβ/δ activity in cultured L6 myotubes treated for 24 h with 0.1 µM GW0742 in the absence or presence of 0.1 µM GSK0660. Results are expressed as percent of control (Ctr). <i>B</i>) FOXO1 mRNA (left panel) and protein levels (right panel) in cultured L6 myotubes after treatment for 24 h with 0.1 µM GW0742. Representative Western blots are shown in the upper and densitometric quantifications in the lower panel. A/C lamin was determined for loading control. <i>C</i>) FOXO1 DNA binding activity in control and GW0742-treated myotubes. <i>D</i>) p-FOXO1(Ser 256) and total FOXO1 protein levels (upper panel) and calculated p-FOXO1/FOXO1 ratio in cultured L6 myotubes after treatment for 24 h with 0.1 µM GW0742. The calculated p-FOXO1/FOXO1 ratio was based on densitometric quantifications of p-FOXO1 and total FOXO1 levels on Western blots. <i>E</i>) Ac-FOXO1 levels in control and GW0742-treated myotubes determined by co-immunoprecipitation. <i>F)</i> Atrogin-1 mRNA and protein levels in cultured L6 myotubes after treatment for 24 h with 0.1 µM GW0742 and in control myotubes. α-Tubulin was determined for loading control. <i>G</i>) MuRF1 mRNA and protein levels in control and GW0742-treated myotubes. <i>H</i>) GW0742-induced increase in mRNA levels for FOXO1, atrogin-1, and MuRF1 in myotubes transfected with a non-targeting (NT) or FOXO1 siRNA. Results are means ± SEM with n = 7 or 8/group. *p<0.05 vs control (Ctr) or NT groups by Student’s t-test (panel B-H) or ANOVA (panel A).</p